Spatial morphological and molecular differences within solid tumors may contribute to the failure of vascular disruptive agent treatments

Treatment of solid tumors with vascular disrupting agent OXi4503 results in over 90% tumor destruction. However, a thin rim of viable cells persists in the tumor periphery following treatment, contributing to subsequent recurrence.

R E S E A R C H A R T I C L E Open Access

Spatial morphological and molecular differences within solid tumors may contribute to the failure

of vascular disruptive agent treatments

Linh Nguyen†, Theodora Fifis*†, Caterina Malcontenti-Wilson, Lie Sam Chan, Patricia Luiza Nunes Costa,

Mehrdad Nikfarjam, Vijayaragavan Muralidharan and Christopher Christophi

Abstract

Background: Treatment of solid tumors with vascular disrupting agent OXi4503 results in over 90% tumor

destruction However, a thin rim of viable cells persists in the tumor periphery following treatment, contributing to subsequent recurrence This study investigates inherent differences in the microenvironment of the tumor

periphery that contribute to treatment resistance

Methods: Using a murine colorectal liver metastases model, spatial morphological and molecular differences within the periphery and the center of the tumor that may account for differences in resistance to OXi4503 treatment were investigated H&E staining and immunostaining were used to examine vessel maturity and stability, hypoxia and HIF1α levels, accumulation of immune cells, expression of proangiogenic factors/receptors (VEGF, TGF-β, b-FGF, and AT1R) and expression of EMT markers (ZEB1, vimentin, E-cadherin andβ-catenin) in the periphery and center of established tumors The effects of OXi4503 on tumor vessels and cell kinetics were also investigated

Results: Significant differences were found between tumor periphery and central regions, including association of the periphery with mature vessels, higher accumulation of immune cells, increased growth factor expression,

minimal levels of hypoxia and increased evidence of EMT OXi4503 treatment resulted in collapse of vessels in the tumor center; however vasculature in the periphery remained patent Similarly, tumor apoptosis and proliferation were differentially modulated between centre and periphery after treatment

Conclusions: The molecular and morphological differences between tumor periphery and center may account for the observed differential resistance to OXi4503 treatment and could provide targets for drug development to

totally eliminate metastases

Keywords: Vascular disruptive agent, OXi4503, Tumor periphery, Hypoxia, Growth factor, Infiltrating cells, EMT

Background

Solid tumors require a well established vasculature to

grow As the tumor grows its vasculature undergoes

con-stant remodeling [1] which makes the tumor

microvascu-lature unstable This characteristic makes the tumor

microvasculature more sensitive to destabilizing drugs

compared to normal host microvasculature Exploiting

these differences to target established tumor

microvascu-lature is a novel concept resulting in the development of

vascular disruptive agents (VDAs) [2] Treatment with VDAs is characterized by rapid and extensive destruction

of tumor limited only by the persistence of a viable rim of tumor in the periphery which subsequently leads to recur-rence [3] The Combretastatins are a family of tubulin binding vascular disrupting agents that specifically target the vascular network within a solid tumor Despite exten-sive tumor destruction, complete tumor eradication is not achieved [4] OXi4503, a derivative of Combretastatin CA4P, is a second generation VDA that is more potent than CA4P, killing more than 90% of tumor [5] It has been shown to be effective in a wide variety of tumor models and is currently undergoing clinical trials

* Correspondence: tfifis@unimelb.edu.au

†Equal contributors

Department of Surgery, University of Melbourne, Austin Health, Heidelberg,

Victoria 3084, Australia

© 2012 Nguyen et al.; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and

(ClinicalTrials.gov Identifier: NCT01085656) Despite its

enhanced potency, treatment with OXi4503 also leaves

the characteristic rim of viable tumor cells albeit smaller

in size than that seen in tumors treated with CA4P [6,7]

As tumor cells survive only in the periphery, we

hypothesize that there are intrinsic differences between

the periphery and the bulk of the tumor that confer

re-sistance to treatment A number of studies reported

increased expression of growth factors in the periphery

[8,9] In a previous study [10] we have shown that

macrophages and T-cells infiltrate the tumor and

prefer-entially accumulate in the periphery Other studies

indi-cate that tumor associated immune cells secrete

cytokines and growth factors that promote tumor

growth [11-14]

The present study examines inherent differences

be-tween the periphery and the bulk of the tumor in a murine

model of colorectal liver metastases including vessel

morphology, immune cell infiltration, expression of

pro-angiogenic factors and markers of Epithelial to

Mesenchy-mal Transition (EMT) Morphological and molecular

changes occurring in the tumor vasculature and in tumor

cell kinetics following administration of OXi4503 are also

investigated

Methods

Animals

Six to eight week old male CBA mice (Laboratory Animal

services, University of Adelaide, South Australia) were

used in all experiments Mice were maintained in standard

cages with access to irradiated food and water ad libitum,

and exposed to a twelve hour light/dark cycle All

proce-dures were implemented in accordance with the

guide-lines of the Austin Health Animal Ethics Committee

Experimental model of colorectal cancer liver metastases

(CRCLM)

The primary cancer cell line MoCR was derived from a

dimethyl hydrazine (DMH)-induced primary colon

car-cinoma in the CBA mouse and maintained in vivo by

serial passage in the flanks of CBA mice [15] For

pas-sage and experimentation, subcutaneous tumors were

teased, passed through a filter, treated with EDTA and

washed in PBS to make a single cell suspension Liver

metastases were induced by intrasplenic injection of

5x104tumor cells prior to splenectomy as reported

pre-viously [15] In this model, liver metastases are fully

established by 21 days following tumor induction The

tumor morphology and growth patterns in this model

have been described previously [6,15,16] Metastases of

varying sizes are found throughout the liver The

metas-tasis pattern is very similar and reproducible within a

group of mice The whole liver is sliced in sections of 2

mm thickness Cross-sections of the larger tumors are

represented in more than one section Random sections are selected to represent the entire liver and used for par-affin embedding and analysis Each section could contain from one to several individual tumors (Additional file 1: Figure S1) Metastases seeded in close proximity often co-alesce into a continuous tumor

Treatment protocol

Treatment was administered sixteen days after induction

of liver metastases when tumors are well established

Inc South San Francisco, CA), was freshly prepared by dissolving in 0.9% sterile saline (NaCl) and protected from light A single maximum tolerated dose of OXi4503, determined previously to be 100 mg/kg [16], was administered via intraperitoneal injection Control groups were administered an equivalent volume of ster-ile saline Tissues were collected at one hour, twenty four hours and five days following OXi4503 treatment

Definition of tumor periphery

Tumor periphery in our studies consisted of the area cov-ering the tumor-host interface and extending one hundred microns towards the tumor center All the remaining tumor area was considered part of the tumor center

Vascular morphology

Vessel morphology was examined microscopically in stained tumor sections Immature vessels and/or vessels undergoing angiogenesis were detected by CD34 staining [17] All CD34 positive vessels/mm2in each tumor sec-tion were counted Vessel stability and maturity were also assessed by pericyte coverage and angiopoetin 1 (Ang1) association [18] The presence of pericytes was visualised byαSMA immunostaining and enumerated by counting of αSMA positive tumor vessels in serial sec-tions stained for αSMA or CD34 Only vessels that stained for both markers were included in the enumer-ation Ang1 association was determined by double immunostaining for Ang1 and CD34

Detection of tumor hypoxia

Pimonidazole was used as a marker of tumor hypoxia Pimonidazole hydrochloride was dissolved into 0.9% NaCl and administered intravenously to tumor-bearing mice in doses of 30 mg/kg The livers were removed one hour after pimonidazole administration and fixed in 10% formalin in 0.1M phosphate buffer, pH 7.2 Hypoxic tumor regions were detected immunohistochemically as reported previously [19]

Assessment of epithelial to mesenchymal transition (EMT)

The main indicators of EMT are down regulation of the cell junction protein E-cadherin, nuclear accumulation

ofβ-catenin another junctional protein, up regulation of

the mesenchymal marker vimentin and up regulation of

transcription inhibitors of epithelial proteins such as

ZEB1 [20,21] The spatial expression of these markers

was assessed for evidence of EMT

Histological assessment

Hematoxylin and eosin (H & E) stained sections were

examined histologically and digital images captured using

a Nikon CoolscopeW (Nikon Corporation, Chiyokd-ku,

Tokyo, Japan) A minimum of 50 tumors were assessed

per treatment group

Immunohistochemistry

Spatial differences in untreated tumors and changes due

to OXi4503 treatment were detected using histological

and immunohistochemical techniques

Antibodies used for infiltrating immune cells; Rabbit

polyclonal antibodies to human CD3 (A0452, DAKO),

Rat anti-mouse monoclonal antibodies to FOXP3

(14-5773-80, e-bioscience), and F4/80 a kind gift from

Pro-fessor Mauro Sandrin Dept of Surgery, University of

Melbourne Antibodies used for growth factor detection;

Rabbit polyclonal antibodies to mouse AT1R (sc-1173),

TGF-β (sc7892), b-FGF (Lot no: 24030710) obtained

from Santa Cruz, VEGF (PC315, CalBiochem) and

HIF1α (AB 3883, Chemicon) Antibodies used for vessel

detection; Rat anti-mouse monoclonal antibodies to

CD34 (MCA18256, Serotec), rabbit polyclonal antibodies

AB, Biocare) and Angiopoetin1 (ab 8451–200, Abcam)

Antibodies used for EMT detection; Rabbit polyclonal

antibodies to mouse E-cadherin 7870), Vimentin

(sc-5568), ZEB1 (sc-25388) and rat anti-mouse monoclonal

antibodies toβ-catenin (sc-7199) all obtained from Santa

Cruz Cell proliferation was detected with rabbit

mono-clonal antibodies to Ki67 (rm-9106-s1 thermo scientific)

and cell apoptosis with rabbit polyclonal antibodies to

Active Caspase-3 (AF835, R&D systems) Additional file

2: Table S1 presents a list of antibody concentrations

and assay conditions used

Formalin fixed paraffin tissue sections (4 μm) were

used with an indirect peroxidase labeling technique

(En-vision Plus, DAKO, Australia) Following

deparaffiniza-tion and rehydradeparaffiniza-tion, endogenous peroxidase activity

was blocked with 3% H2O2 and non-specific binding

inhibited with 10% normal goat serum (01–6201 Zymed

Laboratories, USA) after which epitope retrieval was

conducted (Additional file 2: Table S1) Sections were

incubated with primary antibodies overnight at 4°C

Negative controls were incubated with the respective

non immune antibody isotypes or non-immunized rabbit

IgG (Santa Cruz, sc-2027) at the same concentration as

the primary antibody Sections treated with the rat

antibodies were subsequently treated with a rabbit anti-rat IgG linker antibody before treatment with a polymer based detection kit containing goat anti-rabbit immuno-globulins (IgG) linked to horseradish peroxidase (HRP) (Envision Plus, Dako, Australia) Each incubation step was followed by two five minute washes with PBS + 0.05% Tween 20 Positive staining was visualized using diaminobenzidine (DAB) as a substrate For double immunostaining Vulcan fast red (Applied Medical FR805H) was used to stain CD34 Slides were counter-stained with Mayer’s haematoxylin

A minimum of five mice were used per group and be-tween 75 and 120 tumors were assessed for each time-point/treatment group Images of stained tumors were captured using a digital light microscope (Nikon Cool-scopeW, Nikon Corporation, Japan) at between 10x and 400x magnification The images of tumor fields were captured to be representative of the entire tumor, using

a raster pattern which allowed for fields captured to be random and not overlap Between 10 and 30 fields per tumor (periphery and center) were assessed The images were analyzed using Image-Pro plus (Version 5, Media Cybernetics, Perth Australia) The number of CD34 posi-tively stained vessels per tumor area (mm2) to were counted provide a microvascular density index Ki67, active caspase3, CD3, FOXP3 and F4/80 were assessed as the number of CD34 positive cells per area of tumor (20x mag-nification) Positively stained cells per image were marked and quantification was performed using Image-Pro plus (Version 5, Media Cybernetics, Perth Australia) Differ-ences in hypoxia and the antigens (AT1R, VEGF, b-FGF, TGF-β, HIF1α, E-cadherin, Vimentin, β-catenin and ZEB1) were assessed by microscopic observation and representa-tive images are presented

Quantification of AT1R, VEGF and TGF-β was per-formed using a semi-quantitative analysis Areas of inter-est were identified using a light microscope (Olympus BH2, Japan) at a magnification of 125x The entire margin

of tumor host interface and tumor center were examined Scoring criteria was used to estimate the amount and in-tensity of staining seen in each sample The grading sys-tem used was: as: 0: no staining 1: faint staining; 2: small amount or weak staining; 3: moderate staining; 4: abun-dant or strong staining; 5: Abunabun-dant or very strong stain-ing Means for each group were determined using the individual average scores from each animal in the group For all counting and scoring researchers were blinded in regard to the experimental group

Statistical analysis

Quantified data is represented as the mean ± standard error of the mean Statistical analysis was conducted using SPSS (Statistical Package for the Social Sciences,TM ver-sion 10, Chicago, Illinois, USA) with normality testing and

use of both parametric and non parametric analytical tests

as appropriate All statistical tests were two-sided and a P

value of 0.05 or less was considered statistically significant

Results

Spatial differences in tumor vessel density and vessel

morphology

CD34 and CD31 are two endothelial cell markers often

used in determining tumor vascular density While these

two markers roughly stain the same number of tumor

ves-sels (Additional file 3: Figure S2) neither marker stains all

the tumor vessels In our experience CD34 normally stains

tumor vessels and host vessels undergoing

neovascularisa-tion as seen in liver regeneraneovascularisa-tion (unpublished result) but

stains mature vessels only minimally CD31 shows more

cross-reactivity and also stains liver sinusoids (Additional file 3: Figure S2), therefore in this study we used CD34 Staining and quantification of CD34 positive staining ves-sels (Figure 1A and B) demonstrate significantly stronger staining (Figure 1A inset 1 arrows) and greater density in the central regions of tumor (Figure 1B, P<0.001) Vessels

in the periphery either did not stain or only partially stained with CD34 (Figure 1A inset 2 arrows) Interest-ingly CD34 negative or faintly stained host vessels at the tumor-host interface were seen to be co-opted by tumor cells (Figure 1A inset 3 arrows) Maturity of tumor vessels was assessed by αSMA staining of pericytes associated with the vessels In addition to pericytesαSMA also stains myofibroblasts and in this model there is significant accu-mulation of myofibroblasts within the tumor stroma, es-pecially in the periphery (Additional file 4: Figure S3) To determine pericyte coverage we used serial sections stained for CD34 /αSMA and only vessels that stained for both markers regardless of the strength of CD34 staining were included in the enumeration Tumor periphery showed at least 2.5 times greater pericyte coverage than vessels in the center of the tumor, indicating that the vasculature is more stable and mature in the periphery (Additional file 4: Figure S3 and Figure 1C, P<0.001) Due

to the large number ofαSMA staining myofibroblasts the difference in vessel pericyte coverage is only an estimate and may be underestimated since not all vessels in the periphery stain for CD34 Angiopoetin 1 another vessel stability marker was also preferentially associated with ves-sels in the tumor periphery (Additional file 4: Figure S3) further supporting our finding that the periphery of tumors is associated with relatively mature stable vessels

Spatial differences in the accumulation of immune cells

In a previous study we reported the accumulation of im-mune cells within the tumor [10] In the present study

we demonstrate that accumulation of CD3 T cells, regu-latory T cells and macrophages is significantly higher in the periphery than in central regions of the tumor (Figure 2A and 2B, P values 0.0001, 0.0001 and 0.027 re-spectively) Of particular interest was that regulatory T cells represent a significant fraction (32.4% in the per-iphery and 49.5% in the center) of the T cell population indicating an immunosuppressive function

The periphery of the tumor is normoxic relative to the center

Hypoxia in tumors has been implicated in the develop-ment of resistance to therapy In this study the distribu-tion of hypoxic regions within the tumor were variable and occurred throughout the center Importantly, tumor cells in the periphery were minimally hypoxic Very few cells in the periphery were stained with pimonidazole (Figure 3A first panel and inset 2) except when tumors

Figure 1 Differences in blood vessel morphology between tumor

periphery and center A: Formalin fixed liver sections with CRC liver

metastases were stained with antibodies to CD34 (staining of

endothelial cells on immature vessels) Scale bar=200 μm Inset 1

depicts tumor vessels (arrows) in the tumor center staining strongly for

the CD34 endothelial marker Inset 2 depicts tumor vessels (arrows) in

the tumor periphery staining weakly for the CD34 Inset 3 depicts host

vessels (arrows) being co-opted by the tumor displaying weak or no

CD34 staining B: Quantification of CD34 positive vessels in tumor

center and tumor periphery expressed as CD34 positive vessels /mm 2

Black bars = tumor periphery, Grey bars = tumor center Significantly

more CD34 positive vessels are seen in the tumor center (*P<0.001).

Quantification of αSMA pericyte association with tumor vessels

revealed a significantly greater number in the tumor periphery

compared to the tumor center (* P<0.0001, Black bars = tumor

periphery, Grey bars = tumor center Data is expressed as mean value±

SEM, with n ≥5 for each group Data was not normally distributed and

non-parametric analysis was performed and statistical significance

determined using Kruskal Wallis and Mann-Whitney U test.

were growing on the liver surface Peripheral tumor

regions that did not lay adjacent to liver parenchyma or

host vessels displayed hypoxia, as seen in Figure 3A (first

panel and inset 1) More centrally located tumor cells,

particularly those not in close proximity to major

ves-sels, displayed high levels of hypoxia (Figure 3A) These

results support our observations that the periphery of

the tumor is supplied with mature and stable vessels

HIF1α expression was variable as seen with hypoxia and

displayed a similar distribution pattern, indicating that it

is stabilized by hypoxia, however expression is also seen

in the periphery albeit at lower levels (Figure 3B)

The tumor periphery is associated with upregulated

growth factor expression

Hypoxia and HIF1α are known to stimulate

up-regulation of pro-angiogenic growth factors [22]

Expres-sion of VEGF and the pro-angiogenic receptor AT1R are

markedly up-regulated in the periphery (Figure 3C and

Figure 3D) However, the distribution of VEGF and

AT1R were found to closely mirror the distribution of

infiltrating T cells and macrophages (Figure 2A) rather

than the distribution of hypoxia and HIF1α (Figure 3A

and Figure 3B) This suggests that these factors may be

mainly expressed by or under the influence of the

infil-trating immune cells Similarly b-FGF and TGF-β are

preferentially expressed in the periphery Additionally these two factors are also strongly expressed within the liver parenchyma immediately adjacent to the tumor host interface (Figure 3E and Figure 3F)

The tumor periphery is associated with increased mesenchymal marker expression

The bulk of the tumor cells in CRCLM were found to be strongly positive for E-cadherin and displayed the character-istic cobblestone junctional complex staining (Figure 4A) However, in the periphery a few tumor cells did not express E-cadherin and appeared detached from the main tumor (Figure 4A; inset arrows indicate E-cadherin negative tumor cells) Immunostaining showed that most tumor cells displayed a β-catenin staining pattern similar to that of E-cadherin (Figure 4B) being present mainly in the cell junc-tions In the periphery however the occasional tumor cell was positive for nuclear β-catenin (Figure 4B; inset arrows point atβ-catenin nuclear localization)

Tumors in this study also showed very faint cytoplasmic vimentin staining in the bulk of the tumor Vimentin staining was slightly more intense in the periphery where the occasional tumor cell also displayed nuclear staining (Figure 4C and inset, arrows indicate nuclear vimentin) The majority of tumor cells in our CRCLM tumor model did not express ZEB1 In contrast, strong ZEB1

Figure 2 Preferential accumulation of immune cells in the tumor periphery Formalin fixed liver sections with CRC liver metastases were stained with anti-CD3, anti-FOXP3 and F4/80 monoclonal antibodies to detect the presence of T cells, regulatory T cells and macrophages respectively Low magnification scale bar=250 μm, high magnification scale bar=50μm Quantification of each cell type revealed significant differences between the tumor center and the periphery Data is expressed as mean value of positive cells/ mm 2 ±SEM with n ≥5 for each group (*P=0.0001 for T cells and regulatory T cells and #P=0.021 for macrophages).

staining was seen to be associated with infiltrating stromal

cells that had a mainly fibroblast appearance and

accumu-lated in the tumor-host interface and along major vessels

(Figure 4D) Some of the positive cells had a round

ap-pearance and from their observed location, they may be

mast cells A few ZEB1 positive tumor cells were also

present mainly in the periphery but some also interspersed

throughout the tumor (Figure 4D arrows in inset pointing

at positive tumor cells) Taken together, these results

indi-cate that a proportion of tumor cells in the periphery in

this tumor adopt mesenchymal morphology

Treatment with Oxi4503 results in endothelial cell apoptosis and rapid occlusion of tumor vessels

Having demonstrated several important molecular and morphological differences between the periphery and the rest of the tumor, the differential effect of a single dose of OXi4503 on established CRLCM was then investigated We examined microvascular changes in the tumor center and periphery at one hour, 24 hours and five days following OXi4503 treatment Untreated tumors displayed open functional vessels (Figure 5 con-trol) Within one hour of treatment tumor vessels

Figure 3 Molecular and morphological differences between tumor periphery and center Formalin fixed liver sections with CRC liver metastases were stained for hypoxia by staining for pimonidazole using hypoxiprobe and growth factor/receptor expression HIF1a, VEGF, AT1R , b-FGF and TGF- β using the respective antibodies (A) Low magnification scale bar=500μm, inset magnification scale bar=50μm, Tumors in the periphery show less hypoxia (first row panel 1 and inset 2) unless the tumor periphery lies on the liver surface with no adjacent host tissue (inset 1) (B) HIF1a staining displaying higher staining towards the tumor center in areas associated with high hypoxia (C) VEGF, (D) AT1R, (E) b-FGF and (F) TGF- β, all are expressed at higher levels in the tumor periphery (B-F magnification scale bar=200μm) (G) Quantification of AT1R, VEGF, TGF-β, demonstrate significantly higher staining in the periphery (p<0 0001, p<0 001, and p<0.0001 respectively).

became congested (Figure 5 1hr OXi4503) The

endo-thelial cells lining the vessels appeared rounded and

detached from the vessel wall (Figure 5, 1hr OXi4503

arrows indicate rounding endothelial cells) Using

double staining for active caspase-3 and CD34, we

found that endothelial cells not only changed shape but

also were apoptotic (Figure 5, 1hr OXi4503) At 24

hours, all the central tumor vessels had occluded and

the majority no longer stained with CD34 indicating

endothelial cell death (Figure 5, 24hrs OXi4503, center

and Additional file 5: Figure S4) However, a number of

patent vessels that did not stain or only partially stained

with CD34 were seen in the periphery (Figure 5, 24hrs

OXi4503, periphery and Additional file 5: Figure S4) By

day five, as seen in our previous studies [23] the tumor

had vigorously re-grown towards the necrotic center

and vessels had re-established with increased vessel

density compared with control tumors (Figure 5, 5days

OXi4503, center)

Quantification of vascular endothelial cell apoptosis by

active caspase-3 staining demonstrated that OXi4503

induced significantly more vascular endothelial cell

apop-tosis in the tumor center at one and 24 hours after

treat-ment compared to the periphery (Figure 5B, P <0.001 for

both timepoints) This differential in apoptosis of vascular

endothelial cells resulted in a significant decrease in

vascu-lar density in the tumor center (P<0.0001), but no

significant change in the periphery (P=0.173) at 24 hours after treatment (Figure 5C)

Tumor vessel density in the treated tumor five days after treatment was found to be significantly higher compared to the untreated control both in the bulk of the tumor and in the periphery Vascular density is 1.6 times higher in the periphery and 1.9 times higher in the center of OXi4503 treated tumors compared to controls (Figure 5C, P<0.001 for both) These results demonstrate inherent differential resistance to OXi4503 in tumor vas-culature between the periphery and the bulk of the tumor Furthermore after the initial vessel damage revas-cularization resumed at increased rates indicating that treatment induced signals for angiogenesis

Tumor cells in the periphery are resistant to apoptosis after OXi4503 treatment

Following OXi4503 treatment (Figure 6A) a thin rim of viable cells was seen in H&E stained tumor sections at the tumor host interface at both one hour and 24 hour timepoints No appreciable change in the number of vi-able tumor cells could be seen between these two time-points In control tumors, apoptosis occurs within some central regions but very seldom within the periphery (Figure 6B control) Within one hour following treat-ment, significant apoptosis occurred in the tumor center leading to large necrotic areas (Figure 6B, 1hr OXi4503)

Figure 4 Tumor cells in the periphery express mesenchymal markers Formalin fixed liver sections with CRC liver metastases were stained with antibodies for EMT markers; E-cadherin (A), β-catenin (B), vimentin (C) and ZEB1 (D) Magnification scale bar=200μm Arrows in A magnified inset indicates detached tumor cells not expressing E-cadherin Arrows in B magnified inset indicates tumor cells displaying nuclear localization of β-catenin Magnified inset in C indicates increased vimentin staining in the periphery Magnified inset and arrows in D indicate tumor cells displaying nuclear localization of ZEB1.

This pattern of injury continued at 24 hours (Figure 6B

24 hrs OXi4503) Although apoptosis in the periphery

was significantly increased at one and 24 hours after

OXi4503 compared to controls (Figure 6B graph,

P<0.0001 for both timepoints) there was significantly

lower apoptosis in the treated periphery compared to

center of the treated tumor (Figure 6B graph, P<0.0001

for both timepoints) Furthermore, significantly more

apoptotic cells are seen in the periphery at 24 hours

compared to one hour after treatment, suggesting that

inhibition of apoptotic pathways immediately following

treatment may be part of the resistance mechanism By

five days after treatment apoptosis had virtually ceased

and new tumor growth is seen to extend towards the

center into previously apoptotic areas (Figure 6A and

Figure 6B, 5 days OXi4503)

Tumor cells in the periphery transiently reduce

proliferation after OXi4503 treatment

Quantification of tumor cell proliferation showed that

control tumors exhibited high rates of proliferation in

both the center and the periphery (Figure 6C control) Cell proliferation was drastically reduced both at the center and the periphery at one and 24 hours after treat-ment (Figure 6C, 1 hr and 24 hrs, P<0.001 in all cases compared to untreated control) Comparison of tumor proliferation between periphery and center in treated tumors showed that significantly higher cell proliferation was seen in the periphery at both timepoints (Figure 6C,

1 hr and 24 hrs P<0.0001 for both timepoints) These results show that a significant proportion of tumor cells in the periphery stop proliferating in response to VDA treatment, reaching a minimum at 24 hours after treatment Reduction in proliferation was seen to be only

a transient response however, as by day 5 these cells have recovered and resumed vigorous proliferation (Figure 6A, 5days OXi4503 and 6C, 5days OXi4503) Discussion

Tumor microvasculature unlike that of the host is particu-larly sensitive to vascular disruptive agents such as OXi4503 resulting in rapid vessel thrombosis and

Figure 5 Changes in endothelial cells and vessel morphology following OXi4503 treatment Mice were treated with a single IP dose of OXi4503 (100mg/kg) at 16 days after tumor induction Tissues were collected at one hour, 24 hours and five days after treatment Formalin fixed liver sections were stained with anti-CD34 antibody to visualize tumor vessels (A), Control tumor, arrow indicates a patent tumor vessel; 1hr OXi4503, arrows indicate endothelial cells rounding up and detaching from the vessel basement membrane; 1hr OXi4503, EC apoptosis, the section was doubly immunostained for CD34 and active caspase-3 (apoptosis marker), to visualise endothelial cells undergoing apoptosis (arrows); 24hrs OXi4503 center, arrow points at a totally occluded tumor vessel; 24hrs OXi4503 periphery, arrow indicates patent tumor vessel; 5 days OXi4503 , center, demonstrating regenerating tumor vessels surrounded by proliferating tumor cells; Single staining magnification scale

bar=50 μm, double staining magnification scale bar=25μm (B), Enumeration of vascular endothelial cell apoptosis show significant differences between the tumor center and periphery at one and 24 hours after treatment (*P <0.001); (C), Quantification of tumor vascular changes following OXi4503 treatment Vascular density decreased significantly in the tumor center (**P<0.0001), but not the periphery (P=0.173) at 24 hours after treatment Tumor revascularization at day five is significantly higher compared to the untreated control both at tumor center and the periphery (*P=0.001) Results are mean values ± SEM, (n ≥5) Black bars = tumor periphery, Grey bars = tumor center.

significant tumor cell death A single dose of OXi4503 in

mice at the MTD produces more than 90% necrosis of the

total tumor mass Characteristically complete tumor

eradi-cation is not achieved as a thin rim of viable tumor in the

periphery invariably gives rise to regrowth [6,7,16]

The first part of this study demonstrates several

inher-ent differences between the tumor cinher-enter and periphery

that may account for the differential resistance to VDA treatments

Significantly more tumor vessels stained positive for CD34 in the center of the tumor compared to the per-iphery Tumor vessels in the periphery display greater pericyte coverage and angiopoetin 1 association than vessels in the tumor center In our experience CD34

Figure 6 Changes in tumor kinetics following OXi4503 treatment Mice were treated with a single IP dose of OXi4503 (100mg/kg) at 16 days after tumor induction Tissues were collected at one hour, 24 hours and five days following OXi4503 treatment (A) H&E stained sections at indicated times after OXi4503 treatment Magnification scale bar=200 μm Tumor cells at the live rim at one and 24hrs are seen in the enclosed lined areas indicated by arrows NA= necrotic/apoptotic area, T= tumor, L=liver (B) Tumor cell apoptosis at indicated times after treatment detected by active caspase-3 staining Low magnification scale bar=250 μm, inset magnification scale bar=50μm Graph showing quantification of apoptotic tumor cells Results are mean values ± SEM, (n ≥5) Black bars = tumor periphery, Grey bars = tumor center Apoptosis in the treated tumor periphery was significantly higher than in the periphery of control tumors at one and 24 hours (*P <0.001) but significantly lower than the center of the treated tumors (#P<0.0001) (C) Proliferation changes in tumors at indicated times after treatment detected by Ki-67 staining Low magnification scale bar=250 μm, inset magnification scale bar=50μm Graph showing quantification of Ki-67 positive tumor cells Results are mean values ± SEM, (n ≥5) Black bars = tumor periphery, Grey bars = tumor center Proliferation in the periphery was significantly reduced at one and

24 hours (*P <0.001) following treatment compared to controls Significantly higher number of cells proliferate in the periphery of treated tumors compared to the center (#P<0.0001).

only stains tumor vessels and vessels undergoing

neoangiogenesis as seen in liver regeneration

(unpub-lished data) Our results indicate an inverse

relation-ship between CD34 and αSMA staining The presence

model may have resulted in an underestimation of the

difference in vessel maturity between tumor center and

periphery The lower expression of CD34, greater

pres-ence of pericytes and angiopoietin 1 association in the

periphery suggests significantly greater maturation of

the microvasculature in that region [18] These

find-ings suggest vessels in the center of the tumor are

under constant remodeling while periphery is supplied

by more mature and stable vessels Other differences

between the center and the periphery also include

lower levels of hypoxia in the periphery and

signifi-cantly higher expression of proangiogenic factors

and receptors (VEGF, TGF-β, b-FGF and AT1R) The

relatively stable mature vessels in the periphery and

close proximity to normal host vessels are likely the

reason for minimal hypoxia However, in contrast to

current opinion increased proangiogenic factor

expres-sion, with the exception of HIF1α, does not overlap

with regions of increased hypoxia in our study [22]

Instead we observed a close overlap of increased

proangiogenic factor expression and infiltrating

im-mune cells (macrophages and T-cells) Other studies

have also reported accumulation of infiltrating cells

in-cluding macrophages and T-cells in the periphery of

tumors expressing growth factors and cytokines that

are proangiogenic and cytoprotective to tumor [11-14]

This phenomenon has been noted in both surgically

removed human tumors and in experimental tumor

models including CRC Pro-angiogenic growth factors

such as VEGF in addition to their role in neovascular

formation are also directly cytoprotective to cells

expressing their receptors including endothelial and

tumor cells [24,25] In addition to the growth factors

and cytokines we investigated in this study, there are

several other studies reporting additional pro-tumor

cytokines, enzymes and growth factors being up

regu-lated in the periphery of the tumor [26-31] The tumor

cells at the host interface are morphologically different

and are reported to have undergone EMT, perhaps as

a result of the higher growth factor influence,

confer-ring on them characteristics such as increased invasive

ability and drug resistance [20,21,32-34] In our study

mesenchymal markers ZEB1 and vimentin were

prefer-entially expressed in the tumor periphery, while the

epithelial markers E-cadherin and β-catenin were

reduced from cell junctions of some cells in the

per-iphery, suggesting that these cells have undergone

EMT Our findings in the first part of this study

there-fore demonstrate that the tumor microenvironment in

the periphery is significantly different to that of the rest of the tumor and may account for the differential response to OXi4503 treatment

The second part of our study investigated the effect of OXi4503 treatment on tumor microvasculature and tumor cell kinetics We demonstrated that vessels in the periphery are resistant to OXi4503 as they remain patent following treatment This resistance correlates with the increased vessel maturity and stability in the periphery and spatially overlaps with the observed immune cell ac-cumulation and increased growth factor expression seen

in control tumors Initially it was assumed that tumor cells in the periphery survive VDA treatment due to their close proximity to host vessels [2,35] but recent studies demonstrated retained perfusion within the vi-able rim and patent vessels in the periphery as also seen

in our study [3,7,36,37] However, to our knowledge this study is the first to demonstrate and correlate the matur-ity of the microvasculature in the periphery to its abilmatur-ity

to resist the effects of VDAs A clinical study by Gaya

et al [38] investigating the effect OXi4503 treatment on

a variety of different tumors reported significant increase

in vessel permeability correlated with high expression of angiopoetin 2, a marker of vessel instability [18] While that work involved observations on whole tumor, the re-sult supports our finding that vessel stability correlates with VDA resistance Different types of tumor differ in the degree of vascularization, in their vessel morphology and maturity This variation likely influences the effect-iveness of OXi4503 treatment Wankhede et al [37] showed a mouse mammary carcinoma (4T1) and a human renal cell carcinoma (Caki-1) xenograft were dif-ferentially resistant to OXi4503 treatment when grown

in mouse dorsal window chambers They speculated dif-ferences in microenvironment may account for the ob-servation While the tumor periphery does not fully succumb to the effects of VDA treatments, our study and others have demonstrated that some vessels in the periphery are affected [36,37] Other studies also reported some decrease in perfusion within the viable rim and indications of increased hypoxia [7,36,37] Hyp-oxia is known to inhibit proliferation and indeed our results show significantly reduced proliferation in the periphery after treatment Reduced proliferation was also reported following VDA treatments even when apoptosis was not seen [36,37] We demonstrated that both apop-tosis and proliferation of tumor cells are differentially modulated in the periphery following OXi4503 treat-ment Evasion of apoptosis and temporary inhibition in proliferation are mechanisms adopted in drug resistance [39] Cells with mesenchymal characteristics have migra-tory properties and do not proliferate It is possible that the tumor cells within the periphery are protected by their specific microenvironment, but the stress of the